α-L-Rhamnosidase for obtaining rhamnose, a process for its preparation and its use

ABSTRACT

The invention relates to α-L-rhamnosidase, which catalyzes the cleavage of the bond between terminal rhamnose and the aglycone of rhamnose-containing glycosides, a process for preparing it by biotechnological means, and its use for preparing L(+)-rhamnose (6-deoxy-L-mannose).

The invention relates to α-L-rhamnosidase, which catalyzes the cleavageof the bond between terminal L(+)-rhamnose and the aglycone ofrhamnose-containing glycosides, to a process for its preparation bybiotechnological means, and to its use for preparing L(+)-rhamnose(6-deoxy-L-mannose). In that which follows, this latter sugar isdesignated L-rhamnose.

L-Rhamnose is very well suited for use as a chiral structural componentfor the preparation of different organic compounds. L-Rhamnose or itsderivatives are being used to an ever increasing extent in the synthesisof pharmaceutical products and plant protection agents, as well as inthe spheres of plant and animal cytology, microbiology, immunobiologyand the preparation of fragrances. Thus, using L-rhamnose as thestarting compound, 2,5-dimethyl-4-hydroxy-2,3-dihydrofuran-3-one(Furaneol®) can, for example, be prepared, which, in turn, is used asthe parent substance for various fragrant substances in the foodstuffsand perfume industries.

L-Rhamnose can only be obtained with very great difficulty by thechemical route. It can, however, be isolated extractively from variousnatural sources, for example from the flavone glycosides hesperidin,rutin, naringin and quercitrin, or, for example, from gum arabic ormarine algae, following acid hydrolysis. [Biotechnology andBioengineering, Vol. 33, p. 365 (1989), R. J. Linhardt et al.; EP-A-0317 033; JPA 62293]. Disadvantages of these processes are the elaboratesteps for isolating L-rhamnose, some of which involve using organicsolvents, and, in addition, the aromatic, potentially toxic, by-productsaccruing during the working up, and the constituents of the naturalsources, which constituents fluctuate in composition in dependence onthe seasonal rhythm.

L-Rhamnose can also be prepared in the form of rhamnose-containingheteropolysaccharides by fermentation using bacteria of differentgenera, such as, for example, Alcaligenes, Acinetobacter, Klebsiella,Streptococcus or Lactobacillus. [Enzyme Microb. Technol., Vol. 10, p.198 (1988), M. Graber et al.; J. Amer. Chem. Soc., Vol. 71, p. 4124(1945), F. G. Jarvis and M. J. Johnson; J. Bacteriol., Vol. 68, p. 645(1954), G. Hauser and M. L. Karnovsky].

Disadvantages of these processes are the customarily low yields,conditioned by viscosity, and the necessary separation of L-rhamnosefrom a mixture of different sugars following hydrolyric cleavage of theheteropolysaccharide.

Numerous publications and patents deal with the production ofrhamnolipids by means of fermentation using Pseudomonas aeruginosa.[Applied and Environmental Microbiology, Vol. 51, p. 985 (1986), H.E.Reiling et al.; J. Chem. Techn. Biotechnol., Vol. 45, p. 249 (1989), K.Venkata Ramana et al.; U.S. Pat. No. 4 933 281, Daniels et al.; GermanPublished Patent Application 2 150 375, 1972; U.S. Pat. No. 4 814 272,Wagner et al.]

In principle, four rhamnolipids (RL1-RL4, see FIG. 1) are present in themicroorganism culture solution, which rhamnolipids are composed of oneor two L(+)-rhamnose units and one or two β-hydroxydecanoic acids. [Z.Naturforsch. 40 c, p. 61 (1985), C. Syldatk et al.]. Rhamnolipids 1 and3 are quantitatively the most important.

FIG. 1: Rhamnolipids from microorganisms ##STR1##

In addition, it is known to obtain L-rhamnose from flavone glycosides,rhamnolipids or oligosaccharides by enzymic cleavage using the solubleor immobilized α-Lrhamnosidases naringinase and hesperidinase. [U.S.Pat. No. 5,077,206; Eur. Pat. 88202595.0; Turecek, P. and Pittner, F.,Appl. Biochem. and Biotech. 13, 1-13 (1986)]. Maringinase andhesperidinase have in each case a molecular weight of about 90 kd andwere isolated from Penicillium decumbens and Aspergillus niger,respectively.

Naringinase and hesperidinase catalyze the cleavage of the bond betweentwo monosaccharides, chiefly the elimination of terminal rhamnose fromflavanone glycosides, such as, for example, from hesperidin andnaringin, or from rhamnolipid 3 or 4.

Naringinase and hesperidinase catalyze the cleavage of the bond betweenterminal rhamnose and an aglycone in rhamnose-containing glycosidesappreciably more slowly (factor: 10-100; see Example 1).

The consequence of this is that a very long period of time is requiredin order to completely cleave rhamnose from the rhamnolipid 1-4 mixture,since the rhamnolipids 1 and 3 which are present to the greatest extentquantitatively in the microorganism culture solution are composed ofL-rhamnose and aglycone (fatty acid).

α-L-Rhamnosidase has now been isolated, surprisingly, from Penicilliumsp. which catalyzes the cleavage of the bond between terminal L-rhamnoseand the aglycone of rhamnose-containing glycosides, i.e. which has theopposite specificity to the known α-L-rhamnosidases naringinase andhesperidinase.

The invention thus relates to

1. α-L-rhamnosidase, which can be obtained

by fermenting Penicillium sp. DSM 6825 and/or 6826

separating off the biomass from the culture broth, and

concentrating the culture supernatant.

2. α-L-Rhamnosidase having a molecular weight of 60-100 kd, whichα-L-rhamnosidase contains the amino-terminal amino acid sequence

    D-T-N-D-Q-T-S-A-K-V-D-R-G-T-F-D-D-P-A-A-R-L SEQ ID NO:1

or

    F-F-G-S-X-Q-S-L-Y-L-K-L-V-L-K-F-G-T-L-F-D-X-A SEQ ID NO: 2

and catalyzes the cleavage of the bond between terminal L-rhamnose andthe aglycone of rhamnose-containing glycosides.

3. A process for preparing α-L-rhamnosidase, wherein Penicillium sp. iscultivated in a nutrient medium until α-L-rhamnosidase accumulates inthe culture, the biomass is then separated off from the culture broth,and the culture supernatant thus obtained is concentrated.

4. A use of α-L-rhamnosidase for the preparation of L-rhamnose.

5. Penicillium sp. DSM 6825

6. Penicillium sp. DSM 6826.

The invention is described in detail below, in particular in itspreferred embodiments.

The compound L (+)-rhamnose (=6-deoxy-L-mannose) is designatedL-rhamnose.

A compound, or the moiety of a compound, which does not contain anysugar is designated an aglycone. In this invention, fatty acid compoundsor flavone compounds, in particular, are designated aglycones.

The following abbreviations, which correspond to the single letter codeknown from the specialist literature, are used for the amino acids.

    ______________________________________                                        Amino acid     Abbreviations                                                                            Letter code                                         ______________________________________                                        Glycine        Gly        G                                                   L-alanine      Ala        A                                                   L-valine       Val        V                                                   L-leucine      Leu        L                                                   L-isoleucine   Ile        I                                                   L-phenylalanine                                                                              Phe        F                                                   L-proline      Pro        P                                                   L-serine       Ser        S                                                   L-threonine    Thr        T                                                   L-cysteine     Cys        C                                                   L-methionine   Met        M                                                   L-tryptophan   Trp        W                                                   L-Tyrosine     Tyr        Y                                                   L-asparagine   Asn        N                                                   L-glutamine    Gln        Q                                                   L-asparaginic acid                                                                           Asp        D                                                   L-glutamic acid                                                                              Glu        E                                                   L-lysine       Lys        K                                                   L-arginine     Arg        R                                                   L-histidine    His        H                                                   ______________________________________                                    

α-L-Rhamnosidase can be isolated from the culture broth both in smallquantities (up to 1 gram) and in large quantities (≦1 kg), since thepreparation process can be carried out on a laboratory scale(fermentation of the microorganisms in volumes up to 1 liter) and on anindustrial scale (fermentation of the microorganisms on a cubic meterscale).

The molecular weight of the α-L-rhamnosidase according to the inventionis determined by means of SDS gel electrophoresis (SDS=sodium dodecylsulfate) and by means of gel chromatography. This method of gelchromatography is described, for example, in Molecular Biology of theCell, Bruce Alberts et al., Garland Publishing, Inc. New York & London,3rd Edition, 1983, pp. 174, 265-266.

The abbreviation "IEP" stands for "isoelectric point" and Is defined asthe pH at which the net charge on the protein, or in the present casethe enzyme, is zero. The IEP is determined by means ofchromatofocussing.

Penlcillium sp. was isolated from a compost composed of garden waste in6232 Bad Soden, Germany. The microorganism was isolated and purified inaccordance with processes known to the person skilled in the art byculture dilution and plating out on selective agar. For example, thecompost sample can be suspended in 0.9% strength sodium chloridesolution and an enrichment culture of this suspension can be set up inselective medium containing rhamnolipids and/or rhamnolipid derivatives,preferably C₁ -C₁₈ -alkyl esters of rhamnolipid-2 as the sole carbonsource. C₁ -C₄ -Alkyl esters of rhamnolipid-2, such as, for example,methyl rhamnolipid-2 or tert-butyl rhamnolipid-2, are used withparticular preference as the carbon source. ##STR2##

Penicillium sp. DSM 6825 and DSM 6826 have the following morphologicalcharacteristics (after R.A. Samson et al., Introduction to Food-borneFungi, Institute of the Royal Netherlands Academy of Arts and Sciences,3rd Edition, 1988):

    ______________________________________                                        Penicillium sp. DSM 6825                                                      Branching of the conidia                                                                           monoverticillate                                         Phialides            ampulliform                                              Conidia              spiny                                                    Penicillium sp. DSM 6826                                                      Branching of the conidia                                                                           biverticillate                                           Phialides            flask-shaped                                             Conidia              warty                                                    ______________________________________                                         Penicillium sp. DSM 6825 and 6826 may be fermented together or separately                                                                              

Insofar as they produce the enzyme α-L-rhamnosidase, mutants andvariants can also be employed instead of the isolate DSM 6825 and/or6826. Such mutants can be produced, in a manner known per se, byphysical means, for example irradiation, such as using ultraviolet orX-rays, or with chemical mutagens, such as, for example ethylmethanesulfonate (EMS), 2-hydroxy-4-methoxybenzophenone (MOB) orN-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

The process for preparing the abovementioned α-L-rhamnosidases is asfollows:

Following isolation and purification of Penicillium sp. by repeatedpassage of the mixed culture of the resulting microorganisms inselective medium, an enrichment of the microorganisms which produce theenzyme according to the invention is achieved.

The microorganisms thus obtained are plated out on agar plates(selective medium) in order to obtain pure cultures from the mixedculture.

The pure cultures are replicated and tested for their ability to formthe enzyme according to the invention.

It is found that microorganisms of the genus Penicillium sp. form theenzyme according to the invention. The fungal genus is determined withthe aid of morphological, taxonomic and biochemical criteria inaccordance with methods known to the person skilled in the art. Thecolor of the colonies is green.

The investigations of the ability of the Penicillium sp. colonies toform the enzyme according to the invention lead to the isolation of twostrains which are notable for a particularly high level of production ofα-L-rhamnosidase. These two strains are: Penicillium sp. DSM 6825 andDSM 6826.

In accordance with the rules of the Budapest Treaty, these strains weredeposited with the Deutschen Sammlung von Mikroorganismen undZellkulturen GmbH (German Collection of Microorganisms and CellCultures), Mascheroder Weg 1B, 3400 Braunschweig, Germany, on the 29thNov. 1991 under the numbers: Penicillium sp. DSM 6825 and 6826.

The microorganisms concerned are cultivated under the conditions whichare customary for Penicillium sp. Accordingly, cultivation is effectedon complex or defined media; preferably, the media contain yeastextract, casamino acids, corn steep, meat extract, peptone, caseine,gelatin, tryptone, nitrate, ammonium or urea as the nitrogen source, andstarch, dextrin, sucrose, glucose, glycerol and malt extract as thecarbon source.

Magnesium, calcium, sodium, potassium, iron, zinc, cobalt or phosphatecan be employed as further components. The use of rhamnolipids or theiralkyl esters (crude mixtures or purified rhamnolipids) as the C source,alone or in combination with additional C sources, is found to beparticularly suitable.

The cultivation is effected at 27° C. over a period of 72-120 hr.Isolation of the α-L-rhamnosidase according to the invention, which maybe necessary, is effected in a customary manner, e.g. by filtration orcentrifugation to separate off the biomass, the α-L-rhamnosidase for themost part being located in the supernatant.

The supernatant can be concentrated by ultrafiltration and thenlyophilized. Further purification steps, such as, for example,precipitations, anion exchange chromatography, chromatofocussing, HICchromatography (hydrophobic interaction chromatography), exclusionchromatography and affinity chromatography, may be carried out,depending on the degree of enzyme purity desired.

Preferably, the purification is effected by filtering to separate offthe biomass, subsequent ultrafiltration to concentrate the enzyme in theremaining supernatant, then anion exchange chromatography followed bychromatofocussing, and finally exclusion chromatography.

The α-L-rhamnosidase according to the invention has a molecular weightof 60-100 kD, depending on the degree of glycosilation, and anisoelectric point of 5.6-5.8. The pH optimum for the cleavage ofp-nitrophenyl-α-L-rhamnopyranoside is 5.0-5.5 and the temperatureoptimum is 50°-55° C.

The amino-terminal sequence of the α-L-rhamnosidase from the strainPenicillium sp. DSM 6826 and of naringinase is in principle determinedin accordance with the Edman process, which is known from theliterature. For this purpose, amino acid chains are mixed with phenylisothiocyanate under suitable conditions. The chemical compoundpreferentially attaches itself to the free amino-terminal amino group.In the presence of anhydrous acid, the end terminal amino acid iseliminated as a carbamyl derivative. This compound is investigated inorder to identify the amino-terminal amino acid. The remainder of theamino acid chain (which now lacks the initial amino acid) can now besubjected to renewed treatment with phenyl isothiocyanate to determinethe next amino acid in order, etc. In principle, this procedure can becarried out many times stepwise in succession so that the total aminoacid frequency of the chain can be deduced. In the present case, inaddition to this, the N-terminal amino acid sequencing is carried outusing a gas phase sequencer (type 477 A from Applied Biosystems) and theamino acid analysis using an on-line amino acid analyzer (type 130 A PTCanalyzer from Applied Biosystems). The methods are published in FEBSLetters, Vol. 292, pp. 405-409, 1991.

It was possible to elucidate the following amino-terminal constituentsequence of the α-L-rhamnosidase:

    D-T-N-D-Q-T-S-A-K-V-D-R-G-T-F-D-D-P-A-A-R-L SEQ ID NO:1

or

    F-F-G-S-X-Q-S-L-Y-L-K-L-V-L-K-F-G-T-L-F-D-X-A. SEQ ID NO:2

The α-L-rhamnosidase according to the invention can be employed in freeor in immobilized form, where, in the latter case, all current methodsof immobilization are suitable. Silica gel, for reasons of economy, can,for example, be used as the support.

The α-L-rhamnosidase catalyzes the cleavage of the bond between theterminal L-rhamnose and the aglycone of rhamnose-containing glycosidesand is thus suitable for preparing rhamnose.

The cleavage is carried out in aqueous solutions which are buffered ornot-buffered. Aqueous solutions are, for example, the culture broth ofthe microorganism (no buffering necessary) or distilled water. In thelatter case, a buffering with phosphate or Tris buffer, preferablyammonium acetate buffer, is necessary (concentration of the buffer:5-100 mM, preferably 10-50 mM). The pH of the aqueous solution is pH3.5-8, preferably pH 5-6. The temperature which is necessary for theenzyme activity is between 4° C.-65° C. preferably 45° C.-55° C. Thereaction time depends on the quantities of enzyme and substrate and, toa minor extent, also on the temperature. At a temperature of 45° C.-55°C., the reaction time is 2-24 hours, preferably 5-8 hours. It issensible to arrange a relatively short reaction time at highertemperatures, since the enzyme is more readily degraded. The substratequantity (=quantity of rhamnolipids) which is present in the mixture ismaximally 200 g/l, and the enzyme quantity 0.1-50 U/g of rhamnolipids,preferably 1-10 U/g, and particularly preferably 5 U/g, of rhamnolipids.

Following completion of the reaction, the L-rhamnose is isolated fromthe solution by separating off the fatty phase by means ofcentrifugation or by decanting until a phase separation has beeneffected necessary, the aqueous phase is subsequently clarified, forexample using active charcoal. Clarification is understood to mean theremoval of turbidity substances and coloring substances. This step isadvisable if an L-rhamnose is to be obtained which is as pure aspossible. Subsequently, the aqueous solution is concentrated and theL-rhamnose is crystallized out.

Example 1

Cleavage of the rhamnolipids 1 and 3 by naringinase and hesperidinase

10 g of. rhamnolipid 1 or 3 .are emulsified in 100 ml of ammoniumacetate buffer (50 mM, pH 5.5) or double-distilled water and 150 U ofnaringinase or hesperidinase (from Sigms, Germany) are then added. Thereaction is effected at 70° C. while stirring. The values for which areachieved under these conditions are summarized in Table 1. Under theseconditions, the naringinase cleaves the 10 g of rhamnolipid 3 employedin 4 hours (hesperidinase 7 hours) into 2.5 g of rhamnose (˜98% yield)and rhamnolipid 1. The cleavage of rhamnolipid 1 proceeds appreciablymore slowly (see Table 1) and incompletely, probably owing to theinactivation of the enzyme over the relatively long period of time.

The reaction was monitored using thin layer chromatography, and therhamnose was quantitatively determined by means of HPLC.

    ______________________________________                                        TLC:   Eluent:       CHCl.sub.3 /CH.sub.3 OH/HAc 65:5:2                              TLC plate:    Silica gel 60 F254                                              Spraying reagent:                                                                           MeOH/HAc/H.sub.2 SO.sub.4 conc./anis-                                         aldehyde 85:10:5:1                                              Development:  5 minutes at 120° C.                              ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Enzymic cleavage of rhamnolipid 1 and rhamnolipid 3 by means                  of hesperidinase and naringinase [the V.sub.max values relate to              1 U of α-L-rhamnosidase activity; 1 U is defined as the enzyme          quantity which is capable of cleaving 1 μmol of p-nitrophenyl-             α-L-rhamnopyranoside per min]:                                          Hesperidinase: (Sigma No. H-8137, Penicillium spec.)                          "V.sub.max " rhamnolipid 3:                                                                     ˜50 μg min.sup.-1 u.sup.-1                         "V.sub.max " rhamnolipid 1:                                                                     ˜0.5 μg min.sup.-1 u.sup.-1                        Naringinase: (Sigma No. H-1385, Penicillium decumbens)                        "V.sub.max " rhamnolipid 3:                                                                     ˜80 μg min.sup.-1 u.sup.-1                         "V.sub.max " rhamnolipid 1:                                                                     ˜5.0 μg min.sup.-1 u.sup.-1                        HPLC:  Column:        HPAP (100 × 7.8 mm) Biorad                               Precolumn:     Carbo P (30 × 4.6) Biorad                                Temperature:   85° C.                                                  Eluent:        Double-distilled water                                         Flow rate:     0.4 ml/minute                                                  Load           5 μl                                                        Detector:      Differential refractometer                                                    (Beckmann)                                              ______________________________________                                    

The working up of the rhamnose is effected in accordance with thecustomary methods described in the literature (PCT-EP 91-01426).

Example 2

Cleavage of rhamnolipids 1 and 3 by α-L-rhamnosidase from Penicilliumsp. DSM 6825 and/or 6826

10 g of rhamnolipid 1 or 3 are emulsified in 100 ml of ammonium acetatebuffer (50 mM, pH 5.0) or double-distilled water and 150 U of the enzymeaccording to the invention are then added. The reaction is effected at50° C. while stirring. The values for V_(max) which are achieved underthese conditions are summarized in Table 2. Under these conditions, theα-L-rhamnosidase according to the invention cleaves the 10 g ofrhamnolipid 1 employed in about 5-8 hours (α-L-rhamnosidase fromPenicillium sp. DSM 6825: about 8 hours; α-L-rhamnosidase fromPenicillium sp. DSM 6826: about 5 hours) into 3.05 g of rhamnose (˜94%yield) and the corresponding fatty acid. The cleavage of rhamnolipid 3proceeds appreciably more slowly (see Table 2) and incompletely,probably likewise owing to the in activation of the enzyme over therelatively long period of time.

                  TABLE 2                                                         ______________________________________                                        Enzymic cleavage of the rhamnolipids. The V.sub.max values relate             to 1 U of α-L-rhamnosidase activity; 1 U is defined as the              quantity of enzyme which is able to cleave 1 μmol of p-nitro-              phenyl-α-L-rhamnopyranoside per minute:                                 α-L-Rhamnosidase Penicillium sp. DSM 6825                               from Penicillium                                                                          "V.sub.max " rhamnolipid 3:                                                                  ˜0.03 μg min.sup.-1 u.sup.-1              sp. DSM 6825                                                                              "V.sub.max " rhamnolipid 1:                                                                  ˜40.0 μg min.sup.-1 u.sup.-1              α-L-Rhamnosidase Penicillium sp. DSM 6826                               from Penicillium                                                                          "V.sub.max " rhamnolipid 3:                                                                  ˜0.05 μg min.sup.-1 u.sup.-1              sp. DSM 6826                                                                              "V.sub.max " rhamnolipid 1:                                                                  ˜70.0 μg min.sup.-1 u.sup.-1              The L-rhamnose is once again isolated in accordance with the                  known methods; in addition, the fatty acid which has been                     cleaved off can be isolated by extraction under acid conditions.              ______________________________________                                    

Example 3

Screening for strains which produce α-L-rhamnosidase

Microorganisms were enriched from various soil samples on nutrient mediacontaining the methyl or tert-butyl esters of rhamnolipid 2 as the soleC source using current microbiological methods (Drews, MikrobiologischesPraktikum (Practical Microbiology), 45-84, Springer Verlag 1983), andpure cultures were isolated. Some 37 of the approximately 400 strainswhich were isolated were able to degrade rhamnolipids. However, in onlysome of the strains was it possible to detect significantα-L-rhamnosidase activity; in this context, two strains (Penicillium sp.DSM 6825 and Penicillium sp. DSM 6826) were found to be particularlygood producers.

p-Nitrophenyl-α-L-rhamnopyranoside is used as the substrate fordetecting the α-L-rhamnosidase activity. 10 mg of this substrate aredissolved in 10 ml of ammonium acetate buffer (pH 5.5, 50 mM). 100 μl ofculture filtrate or cell lysates are added to 900 μl of this solutionand the mixture is then incubated at 40° C. After 0, 3, 6, 9 and 12minutes, 200 μl are removed and mixed with 800 μl of 200 mM boratebuffer, pH 9. The release-of p-nitrophenol is monitored photometricallyat 410 nm; naringinase (following Romero et al. Anal. Biochem. 149566-571 (1985)) was used as the control.

Example 4

Production of the α-L-rhamnosidases using the strains Penicillium sp.DSM 6825 and Penicillium sp. DSM 6826

These strains are first of all streaked out on agar plates containing HAmedium (yeast extract 4 g/l, malt extract 10 g/l, glucose 4 g/l, agar 20g/l, pH 6.0), and the plates are incubated at 25° C. for 10-14 daysuntil good sporulation has been achieved. A spore suspension (50 ml 0.9%NaCl;0.05% Tween 80) is prepared from two plates which are well grownover and then used for inoculating 10 l of production medium.

The following nutrient solution is used as the production medium: 3 g/1of rhamnolipid 1 or 2, or alkyl esters of the rhamnolipids, orrhamnolipid mixtures (e.g. concentrated filtrate from Pseudomonasaeruginosa culture), 1 g/l KH₂ PO₄, 0.5 g/l (NH₄)₂ SO₄, 0.1 g/lMgSO₄.7H₂ O, 0.1 g/l CaCl₂, 0.1 g/l casamino acids, pH 5.5. Cultivationis carried out in a 10 l paddle mixer reactor at 300 rpm and 0.6 vvm,and at 27° C. and a pH of 5.5. The period of cultivation is 5-10 days.

At the end of the fermentation, the cells are separated off byfiltration and the culture filtrate is filtered (0.22 μm) to render itsterile. This culture filtrate contains the major portion (over 90%) ofthe α-L-rhamnosidase activity (5000 U/l) and can be employed eitherdirectly or following lyophilization, or concentration byultrafiltration on a 10 kD membrane, for cleaving the rhamnolipids.

Example 5

Isolation and characterization of the α-L-rhamnosidase from Penicilliumsp. DSM 6826

A concentrate possessing an activity of 50 U/ml was used as the startingmaterial for the further purification of the α-L-rhamnosidase.

Chromatography on Sepharose Q containing 20 mM Tris/HCl, pH 7.6, iscarried out as the first step. Elution is effected using a gradient of0-0.5M NaCl and the yield which is obtained is in the region of 80%, inassociation with a purification factor of 5; the enzyme possesses aspecific activity of 62 U/mg of protein.

This fraction is subjected to further chromatography on a Mono P column(Pharmacia) (25 mM imidazole/HCl to PBE 74, pH 5.0, 1:12). Theα-L-rhamnosidase is eluted at a pH of 5.6-5.8. The yield is in the orderof 70%.

Following appropriate rebuffering, the protein is chromatographed on aSuperose 12 column (1×30 cm). 100 mM ammonium acetate, pH 5.0,containing 100 mM NaCl is used as the buffer. Monitoring with the aid ofSDS gel electrophoresis indicates a protein band in the range from60-100 kd, depending on the extent to which the enzyme is glycosylated.

The investigations summarized in the table below were carried out usingthe purified enzyme.

                  TABLE 3                                                         ______________________________________                                        Influence of different substances on the activity of the                      α-L-rhamnosidase according to the invention                             Substance           % activity                                                ______________________________________                                        Control             100                                                       CaCl.sub.2 (20 mM)  127                                                       MgSO.sub.4 (2 mM)   106                                                       KCl (100 mM)        69                                                        CsCl (2 mM)         96                                                        CoCl.sub.2 (2 mM)   41                                                        CuCl.sub.2 (0.5 mM) 40                                                        FeSO.sub.4 (0.5 mM) 100                                                       MnCl.sub.2 (2 mM)   27                                                        ZnCl.sub.2 (2 mM)   65                                                        EDTA (10 mM)        22                                                        EDTA/CaCl.sub.2 (10/10 mM)                                                                        90                                                        EGTA (10 mM)        60                                                        EGTA/MgSO.sub.4 (10/10 mM)                                                                        124                                                       L-Rhamnose (0.5 M)  51                                                        L-Rhamnose (1.0 M)    39.5                                                    L-Rhamnose (1.5 M)    26.4                                                    ______________________________________                                    

Example 6

Determination of the N-terminal sequence of the α-L-rhamnosidase fromthe strain Penicillium sp. DSM 6826, and of naringinase

The enzyme from Penicillium sp. DSM 6826, which enzyme was obtained inaccordance with Example 5, as well as the naringinase from Penicilliumdecumbens (crude enzyme, Sigma No. N-1385), which was purified by thesame process, are purified again on an acrylamide gel (10%) and,following transfer of the polypeptides by means of electroblottingtransferred to a ProBlot® membrane (from Applied Biosystems, SequencersNo. 42, April 1990, (Applied Biosystems). Under these conditions, thenaringinase gave rise to a single band whereas the purified enzyme fromDSM 6826 could be resolved into two bands which were very closelyadjacent to each other. The N-terminal-sequences of the threepolypeptide chains are determined with the aid of a 477a PeptideSequencer from Applied Biosystems. The results in Table 4 demonstrateclearly that both the polypeptide chains of the active preparation fromstrain DSM 6826 are different from naringinase (Sigma No. N-1385).

                                      TABLE 4                                     __________________________________________________________________________    Peptides    N-terminal sequence                                               __________________________________________________________________________    Naringinase (96 000 D)                                                                    A S V P X G E X I L A P S S I E L I P T                                                                  SEQ ID NO: 3                           α-L-rhamnosidase 6826,                                                              D T N D Q T S A K V D R G T F D D P A A R L                                                              SEQ ID NO: 1                           peptide I (96 000 D)                                                          α-L-Rhamnosidase 6826,                                                              F F G S X.sub.1 Q S L Y L K L V L K F G T L F D (X.sub.2)                                                SEQ ID NO: 2                           peptide II (83 500 D)                                                         __________________________________________________________________________     X.sub.1 denotes probably cysteine                                             X.sub.2 amino acid not determined                                        

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspThrAs nAspGlnThrSerAlaLysValAspArgGlyThrPheAsp                             151015                                                                        AspProAlaAlaArgLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="Xaa is probably cysteine."                      (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 22                                                               (D) OTHER INFORMATION: /note="Xaa is an undetermined                         amino acid."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PhePheGlySerXaaGlnSerLeuTyrLeuLysLeuValLeuLysPhe                              151015                                                                         GlyThrLeuPheAspXaaAla                                                        20                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="Xaa is an undetermined                          amino acid."                                                                  (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /note="Xaa is an undetermined                          amino acid."                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaSerValProXaaGlyGluXaaIl eLeuAlaProSerSerIleGlu                             151015                                                                        LeuIleProThr                                                                  20                                                                        

We claim:
 1. A purified culture of Penicillium sp. DSM
 6825. 2. Apurified culture of Penicillium sp. DSM
 6826. 3. A purified culture of amutant of Penicillium sp DSM 6825, wherein said mutant produces anα-L-rhamnosidase that catalyzes the cleavage of the bond betweenterminal L-rhamnose and the aglycone of a rhamnose-containing glycoside.4. A purified culture of a mutant of Penicillium sp. DSM 6826, whereinsaid mutant produces an α-L-rhamnosidase that catalyzes the cleavage ofthe bond between terminal L-rhamnose and the aglycone of arhamnose-containing glycoside.
 5. A process for preparing anα-L-rhamnosidase that catalyzes the cleavage of the bond betweenterminal L-rhamnose and the aglycone of a rhamnose-containing glycoside,comprising the steps ofcultivating a Penicillium in a nutrient mediumuntil α-L-rhamnosidase accumulates in the medium;wherein saidPenicillium is selected from the group consisting of Penicillium sp. DSM6825, Penicillium sp. DSM 6826, a mutant of Penicillium sp. DSM 6825that produces said α-L-rhamnosidase, and a mutant of Penicillium sp. DSM6825 that produces said α-L rhamnosidase; separating the biomass fromthe culture medium; and concentrating the culture-supernatant thusobtained.